University of Ljubljana Faculty of Mathematics and Physics. Department of Physics. Seminar I a - 1st year, 2nd cycle.

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1 University of Ljubljana Faculty of Mathematics and Physics Department of Physics Seminar I a - 1st year, 2nd cycle STED Microscopy Author: Nika Mlinari Advisor: prof. dr. Igor Mu²evi Ljubljana, January 2016 Abstract Stimulated emission depletion (STED) microscopy is a scanning far-eld uorescent microscopy technique with sub wavelength resolution. This seminar presents the basic principles of the technique with the focus on uorescence, stimulated emission, excitation and STED beam, setup of a STED microscope and modied resolution equation. In addition, confocal microscopy is briey explained, because it is commonly used to compare STED and confocal images. The second part of the seminar is focused on improved STED variations and STED reasearch in Slovenia.

2 Contents 1 Introduction 1 2 Basic principles Confocal microscopy Fluorescence and stimulated emission Excitation and STED beam STED setup Resolution Improved variations and research T-Rex STED Supercontinuum laser source STED Fast CW-STED Multi-lifetime multi-colour STED STED in Slovenia Conclusion Introduction Resolution of a microscope tells us the minimum distance between two entities in the sample that are seen as separate entities in the image plane. The image of a point in the sample is not a point or a well dened disc, but a series of blurred discs called Airy discs. [1] That is due to diraction and nite dimensions of optical elements. The minimum distance between two separate points is dened as a distance, where the maximum of the rst Airy function falls in the rst minimum of the second Airy function. Ernst Abbe found in 1873 that light with a wavelength λ, travelling in a medium with refractive index n and converging to a spot with angle α will make a spot with radius r = λ 2n sin α where n sin α NA, numerical aperture. Its values are around in modern optics. This formula is known as Abbe diraction limit. All far eld optical imaging systems are limited by diraction limit. It's form may vary from Abbe's in a numerical factor, due to geometry of a system. Let us set up a Cartesian coordinate system with z axes parallel to the optical axes of a microscope. This coordinate system will be used for the whole seminar. Spatial resolution limit of all far-eld light microscopes in 20. century is [2] d xy 200 nm in the sample plane (xy plane). The axial resolution of modern optical microscopes is d z 500 nm. One way to achieve sub wavelength resolution with optical microscope is near-eld optical microscopy, where interactions between light and sample are conned to a small space using a sharp tip. [3] Another way is to use uorophores as a light source. Their manipulation allows sub-diraction resolution in far-eld microscopy. One example of such technique is STED microscopy. 1 (1)

3 Stimulated emission depletion (STED) microscopy is a form of scanning far-eld uorescence microscopy. [2] Two laser beams are used, rst to excite uorescent dye, and second doughnutshaped to cause stimulated emission in the outer parts of the excited region. Photons emitted from the sample through stimulated emission have the same direction as the photons that stimulated the emission - away from the detector. Fluorescence that reaches the detector therefore only comes from the spontaneous emission photons from the central region, that was not depleted through stimulated emission. The dimensions of this central region may be smaller than wavelength of visible light, thus resolution of such microscope is sub wavelength ( 20 nm [4] ). This technique was rst proposed in 1994 by Stefan W. Hell and Jan Wichmann [5] and rst experimentally demonstrated in 1999 by Thomas A. Klar and Stefan W. Hell. [3] In 2014 Hell was awarded Nobel prize together with Eric Betzig and William E. Moerner "for the development of super-resolved uorescence microscopy". [6] 2. Basic principles The rst focus of this section is confocal microscopy which is closely related to STED. Next, principles of STED mycroscopy are discussed in detail. 2.1 Confocal microscopy I would like to say a few words about confocal microscopy before we focus exclusively on STED. The rst reason for this is that STED images are commonly compared with confocal images to show improvement in resolution. The second reason is that STED and confocal principle can be combined, as was the case with rst experimental demonstration of STED by Hell and Klar in [3] Confocal microscopy is a scanning optical microscopy technique that has high classical resolution. It gives us 2D and 3D images, is relatively fast and commonly used. It's spatial resolution is dened by [7] d xy 0.4λ NA and is slightly better than resolution of a standard microscope. The axial resolution is (2) d z 1.4nλ NA 2 (3) where n is the refractive index of imaging medium. [7] In practise, that means that spatial resolution is around 200 nm and axial around 300 nm. This is achieved by adding pinholes at the illumination and detection plane as shown in gure 1. Light that comes from the focal plane in observed sample is focused on the pinhole of the size of a diraction-limited spot (equation (1)) and reaches the detector. Light from the parts of the sample that are out of focus is not focused directly on the hole and is in large part blocked by the aperture. Confocal microscope therefore suppresses the light from out-of-focal-plane and improves the signal to noise ratio. Confocal detection can be implemented in STED microscope. [8] The basic way of scanning the sample plane is in lines. The fast axes is along the lines and the slow axes perpendicular to the lines. The scanning is performed with galvano mirrors, 2

4 acusto-optic deectors or resonant scanners. The deections along the z axes are controlled by a piezo-stage that moves the objective lens. Figure 1: Setup of a confocal microscope. 2.2 Fluorescence and stimulated emission Fluorescence is a property of some atoms and molecules called uorophores. They absorb electromagnetic radiation which excites electrons from ground state to higher excited states. When electrons transition back to the ground state, photons of light with longer wavelength are emitted. Fluorescence is commonly used in far eld microscopy. It allows observation of biological specimens marked with uorescent dyes. Figure 2 shows energy levels of a typical uorophore. [5, 7] S 0 and S 1 are the ground and rst excited electron states and L i mark vibrational levels of S 0 and S 1 that are involved in excitation and emission process. Fluorophore is excited with photons with frequency ν ex. Electrons transition from L 0 level of ground state to L 1 level in excited state. Time scale for this transition is fs. Next electrons relax to state L 2 through vibration on a timescale of ps. L 2 is a singlet state. Transition from L 2 to L 3 in ground state is allowed through radiation and has a characteristic time on a scale ns. During this transition a photon of ν em < ν ex is emitted. L 3 to L 0 transition is again through vibration. [7] Exact positions of the levels of course depend on a specic uorophore, but the principle is the same. Described cycle is shown in the left scheme of gure 2. We can write the following relation between the excitation and emission intensities I em = QσI ex (4) where Q denotes quantum eciency and σ cross section for the absorption. Photons emitted through spontaneous emission have random directions, therefore uorescence is isotropic. 3

5 Figure 2: Energy levels of a typical uorophore. Left scheme shows excitation and spontaneous emission (uorescence). Right scheme shows excitation and stimulated emission. Right scheme of gure 2 shows another process uorophores can undergo. It again starts with absorption of a photon that leads to electron excitation from state L 0 to L 1. Electron relaxes through vibration to state L 2, same as before. At this point, incident photon (left red photon of gure 2) arrives at the sample and interacts with the excited electron, causing it to drop to a lower energy state (in our case L 4 ). In order for this to happen, the incident's photon's frequency must be such that it corresponds to the transition, that is allowed by radiation, in our case ν ST ED = E(L 2) E(L 4 ) h where h is the Planck constant and E energy. During this transition, another photon of ν ST ED is emitted. This photon has the same phase, frequency, polarization and direction as the incident photon. This type of emission is called stimulated emission. Both described cycles are combined in STED microscopy. First, the sample is exposed to the excitation pulse with ν ex that induces the L 0 to L 1 transition in the irradiated region. Next, the second laser pulse with ν ST ED arrives at the sample and causes stimulated emission with electron transitions from L 2 to L 4. The pulses have dierent spatial proles, so only part of the region, excited by the rst pulse (usually outer part) is 'switched o' by the second. This depletion happens before uorescence takes place. [5] Both laser pulses are discussed in detail in the next subsection. The nal step of the process is uorescence from a non-depleted region with the frequency of ν em. It is isotropic and part of it is detected by detector. In 1999, when STED was rst utilised, STED pulse operated at the same frequency as spontaneous emission. [3] Stimulated depletion and uorescence were therefore separated temporarily and through direction of the photons (uorescence is isotropic and stimulated emission photons have the same direction as incidence photon) but had same frequencies. As STED evolved stimulated emission employed dierent energy levels, [7] in our case level L 2 to L 4 in ground state. Level L 4 has greater energy than level L 3, so the emitted photons with frequency ν ST ED are red-shifted and can be distinguished from uorescence photons, ν ST ED < ν em. We can write a system of dierential equations for population probabilities n i (t) of the levels L i. They include absorption, vibrational relaxation, quenching, stimulated and spontaneous emission. [5] (5) 4

6 dn 0 = h ex σ 01 (n 1 n 0 ) + 1 (n 3 + n 4 ) dt τ vibr dn 1 = h ex σ 01 (n 0 n 1 ) 1 n 1 dt τ vibr dn 2 = 1 ( ) 1 n 1 + h ST ED σ 24 (n 4 n 2 ) + q n 2 dt τ vibr τ fluor ( ) dn 3 1 = + q n 2 1 n 3 dt τ fluor τ vibr dn 4 dt = h ST ED (n 2 n 4 ) 1 τ vibr n 4 (6) where τ fluor is the average uorescence lifetime, τ vibr is the average vibrational relaxation time (with assumption that all vibrational relaxations in our system have similar characteristic times), σ i are the corresponding cross sections for absorption (typical values ) and h i photon uxes ( 10 MW/cm 2 ). Parameter q denotes quenching and it's typical values are around 10 8 s 1. Above equations do not include uorescence reduction due to long-lived triplet states and photobleaching, a photocemical alteration of a uorophore molecule so it is permanently unable to uoresce. Photobleaching increases with intensity of laser pulses. Both eects depend on a uorophore dye we use. 2.3 Excitation and STED beam We have already established that we need two states, a uorescent (on) state and a dark (o) state. They are connected by a transition that can be induced by light. The probability that the molecule remains in the initial state after being exposed to laser beam decreases exponentially with the beam intensity I as e I/Is. Saturation intensity I s is a characteristic of the transition used, scaling inversely with the lifetime of the two states. If we apply I > I s, the probability for transition is more than 63%. If I > 5I s, the transition is almost certain with more than 99% probability. The uorescent state has nanoseconds-long lifetime, which corresponds to I ST ED 5 MW cm 2 for stimulated emission. Such intensities are best achieved with spot scanning. [2] The rst beam, used for excitation has Gaussian intensity distribution in the focal plane. The ideal shape for the second, STED beam is [9] { 0 in the coordinate origin I = 0 else In practice, the STED pulse is doughnut-shaped Gauss-Laguerre beam. (7) In STED, sample is rst exposed to the Gaussian excitation beam. The second, STED beam arrives next and causes stimulated depletion. Both pulses are co-aligned but the beams have dierent spatial proles in the sample plane. Figure 3 shows the shape of the excitation pulse (top), STED pulse (middle) and the region that is not depleted by the STED pulse and uoresces (bottom). Both pulses need to be timed perfectly - STED pulse arrives few ps after the excitation pulse, [3] so the electrons are already relaxed from state L 1 to L 2. The optimal temporal delay is determined experimentally to ensure that the depletion of uorophores in the outer region is maximal. Both pulses take place before uorescence. Right side of gure 3 shows the temporal scheme of STED. The upper part of the gure depicts the case, where 5

7 there is no stimulated emission and the entire excited region uoresces. The lower case shows STED, with excitation and depletion pulse. Fluorescence signal is is lower, because the region that uoresces is smaller than in the rst case, as STED pulse depleted outer parts of the excited region. Figure 3: Left: The shape of excitation beam (top), STED beam (middle) and remaining uorescent region (bottom). Source: [11]. Right: Temporal scheme of excitation and uorescence (top) compared with STED (bottom). Courtesy of M. Vitek. Gauss-Laguerre STED beam does not have so well-dened hole in the middle as the beam from equation (7). If the intensity in the centre is not 0, some electrons in the central point are depleted to ground state. This means a reduction in maximal signal strength. [5] 2.4 STED setup Figure 4 shows a basic STED setup. We see two separate light sources, one for excitation and one, red shifted, for STED beam. In practice both beams can be obtained from one laser source by utilisation of lters or frequency doubling. The second approach was taken by Hell and Klar in I would like to give some numbers tu illustrate the physics behind the setup, so I decided to focuse on a setup from 1999 metioned above. [3] The laser used was mode-locked Ti:sapphire laser. The basic frequency corresponding to 766 nm was used for STED while excitation beam was frequency doubled at 383 nm. The excitation pulse had a duration of 140 fs, which is up to 10 times shorter than the vibrational relaxation and times shorter than uorescence relaxation of the uorophore used in the experiment. It's power was 2µW and well below saturation. The second, STED pulse was delayed ( ps) and its length was around 50 ps. It's power was 28.3 mw. Powers of excitation and STED beam are dierent by a few orders of magnitude. That is because the probability for depletion depend exponentially on the applied intensity, as described in the previous subsection. The purpose of λ/2 phase plate in STED beam is to make sure both excitation and STED beam have the same polarization. Beams are recombined using dichroic mirrors. Our sample can be seen on the right side of gure 4. After the excitation and stimulated emission, uorophores in the small central region relax to ground state through uorescence. Back-emitted uorescence travels towards left side of gure 4 through a dichroic lter to separate it from scattered photons of the excitation and STED beam. The passband of a lter from 1999 experiment was nm. The last thing to do is light detection, which can be done with avalance photodiode or CCD detector. 6

8 2.5 Resolution Figure 4: Setup of a STED microscope. Overcoming the resolution diraction-barrier in far-eld microscopy inspired a new name for super resolution microscopy techniques: optical nanoscopy. The resolution of STED nanoscopy with a doughnut-shaped Gauss-Laguerre beam is given by equation [8] d xy λ 2NA 1 + I/I s (8) where λ is the wavelength of light, NA is numerical aperture of objective lens, I is the maximum intensity of the STED beam and I s saturation intensity characteristic of the dye. We get diraction limit for I = 0. A more appealing limit is I with theoretical spatial resolution d = 0 as the size of the central hole decreases with I as shown in gure 5. The basic STED as described above does not improve axial resolution. This can be achieved with specially designed beams. Figure 6 shows comparison between STED and confocal image. Figure 5: We see the cross-section of the STED beam in the sample plane where r is the distance from the coordinate origin, w is beam width, I intensity and I S saturation intensity. First we take a beam with peak intensity I 1 (blue line). The molecules in the region where I > 5I S are depleted with 99 % probability and we get a central non depleted spot. Next, we take a beam with higher peak intensity, I 2 > I 1. The central, non depleted region is smaller than before. 7

9 Figure 6: STED image of actin laments - scale bar 5 µm (left), comparison of confocal and STED image (center) and comparison of FWHM of confocal and STED (right). Source: [14] 3. Improved variations and research STED microscopy is still evolving and there are many tweaks that improve it's performance. In this section, I focused on four dierent improvements. The last subsection presents STED in Slovenia. 3.1 T-Rex STED Excitation and STED pulse may excite electrons from singlet state S 1 to higher excited states, for example triplet state. This slate is relatively long-lived and dark, meaning that we get no uorescence from the transition from triplet state to lower energy state and therefore lose signal. The idea of T-Rex STED is simple: time between two consecutive pulse pairs is large enough to allow electron relaxation from triplet state. It's average lifetime is in microsecond range, therefore suitable pulse repetition rate is <1 MHz. T-Rex technique alleviates uorescence reduction of dyes that have signicant dark state build-up and also allows higher I/I s ratios and therefore improves resolution. [10] 3.2 Supercontinuum laser source STED Supercontinuum laser is a laser with a very broad spectral bandwidth ( nm). It was rst used in STED microscopy in 2008 by Hell and his colleagues [10] as supercontinuum lasers reached spectral power densities of 1 mw/nm and more. In other words they became powerful enough for STED. Moreover their repetition rate was around 1 MHz which is suitable for T-Rex STED. T-Rex STED was previously implemented with complicated and expensive (half a million US dollars) custom made systems. The advantages of commercial supercontinuum systems is their price (about one tenth of previous systems) and straightforward T-Rex implementation. Excitation and STED pulse originate from the same resonator and are therefore synchronised. In 2008 experiment, excitation beam frequencies were extracted with interference bandpass lters. The STED beam frequencies were selected with a highly dispersive Brewster prism and a mechanical slit. Position and width of the slit dened the spectrum. The setup allowed optimization of the STED spectrum while the measurements were running. This makes it easy to use dierent uorophores in consecutive experiments. The adjustable width of STED spectrum also allows larger I/I s ratios, but we must keep in mind, that the useful bandwidth of STED is around 20 nm for most uorophores. 8

10 The broad range of frequencies also means that more uorophore dyes became suitable for STED microscopy. Described experiment was the rst ever to report STED at 700 nm. [10] 3.3 Fast CW-STED The main advantage of this STED implementation is it's speed. Continuous-wave lasers allow fast scanning with both excitation and STED beam illuminating the sample simultaneously and continuously. I will focus on an experiment from 2010 when CW laser was rst used for STED microscopy. It was carried out by Hell and his colleagues. [8] They used Argon laser (488 nm) for the excitation and two continuous-wave ber-lasers (592 nm) for STED. Each CW provided 1 W and they had to be combined in order to achieve power required for stimulated depletion. They used 15 khz resonant scanning mirror for the fast axis and a piezo-stage for the slow axis. Initially they tested the performance of the described setup and it's resolution. They tested 5 commonly used uorescent markers to demonstrate the high potential of 592 nm as a CW- STED wavelength of choice for biological investigation. They determined that the optical resolution in their experiments was better than 60 nm. Next, they focused on the mycroscopy of the endoplasmatic reticulum of a living cell with the yellow uorescent protein Citrine. Citrine was at that point the best uorescent protein tested for STED in the visible range. [8] They recorded 100 images with the size of 12 µm 6 µm with the pixel size of 25 nm. Each one was acquired in 190 ms with a dwell-time of 0.94 µs. The setup can record images without brakes in between, but in described experiment, the interval between two consecutive frames was 1.5 s in order to see signicant displacements of the structure. The optical resolution of this images is slightly lower than in xed samples but it is still better than 65 nm. The CW-STED does not require beam synchronization, pulse length optimization or delays between beams. The average power of the STED beam used in CW mode is signicantly higher than in pulsed STED system, but in the same time focal peak power is lower by about the same factor. Still, when operating CW-STED at high power levels one must consider that living cells may be damaged by the laser. Another problem is optical trapping that might occur, although the doughnut shape and size are usually unfavourable for this phenomena. [8] 3.4 Multi-lifetime multi-colour STED This subsection is based on a 2011 experiment by Hell and his colleagues. [4] The motivation for this implementation is colocalization analyses where the interplay between two or more proteins is studied in cellular environment. The common way to do it is to use dierent uorescent markers for dierent proteins. Such studies are usually performed with confocal microscope. If observed objects are spheres, their centres can be calculated down to a few nanometers and the resolution of confocal microscope is sucient. The problem arises if the objects are arbitrarily shaped. The degree of protein colocalization is dened by the overlap of uorescence between separate color channels, but the reality is that the two features may overlap in the confocal image and still be separated in real space. A better resolution technique would give better results and that is why STED came in to play. Special attention must be paid to the design of the STED microscope and experiment in order to get nanometer accuracy. The acquisition of the images should be fast and color channels should be recorded in parallel to minimize the inuence of thermal drift and mechanical creeping. This ensures that closely spaced objects are recorded within a short time interval which is particularly important when imaging living specimens. 9

11 As stated in the rst paragraph, colocalization requires two or more uorescent dyes that can be distinguished from one another. The rst way to do this is by using two dyes that emit at dierent wavelengths. This is complex, because there are multiple wavelengths in play: excitation, STED and uorescence of both dyes. Spectrum of mentioned processes are continuous so the overlaps need to be taken in account. It is important not to excite the longer wavelength dye with the shorter wavelength STED pulse. The second way to observe dierently marked objects is to use two dyes that absorb and emit at approximately the same wavelength and have dierent life times. In this case both dyes can be excited and depleted with the same pair of beams and the uorescence signals are temporarily separated. I will describe both options in the following paragraphs. Let us start with multi-lifetime STED. The typical lifetime of organic uorophores are 2-5 ns. If the two dyes are selected one from the lower and one from the upper end of this range, the dierences are usually sucient to discriminate them by lifetime. As said before, dyes are excited and depleted by the same pair of pulses. Fluorescence of both is detected by the same detector and photons are assigned to a dye according to the time of their detection. The next step is to nd a suitable model for uorescence decay and use mathematical algorithms for discrimination. The multi-colour STED needs more pulses and a careful selection of uorophore dyes. Ideally, beams are perfectly aligned. In practice, focal points my be separated due to dierent wavelengths and imperfections of optical elements. If the oset is constant (and in the case of rigidly mounted lens it is) the images of the color channels can be shifted accordingly. The separation between the two dyes can be done based on their emission spectra with a dichroic beam splitter in the detection path. Each channel is detected with a separate detection unit. This simple scheme is not perfect, because the absorption and uorescence spectra usually overlap and some uorescence leaks to another channel. Spectral separation can be improved with more complex experiment setup and linear unmixing. Multi-lifetime and multi-colour STED can be combined to provide means for colocalization with up to three dierent colours. [4] This method was used in 2011 experiment to observe proteins lamin, tubulin and clathrin in human glioblastoma cells. The dyes with similar spectral properties and dierent lifetimes were far-red dyes KK 114 (lifetime (3.1 ± 0,5) ns) and ATTO 647N (lifetime (1.8 ± 0.6) ns) and the third dye with a different spectral properties was ATTO 590. [4] They used a single supercontinuum laser source for all four pulses (2 excitation and 2 STED pulses). 3.5 STED in Slovenia There are currently two STED setups in Slovenia. One is located in the Department of condensed matter physics at the Joºef Stefan institute and one at the Faculty of medicine. The STED system at the Faculty of medicine was set up 2 years ago in cooperation with professor Hell and his colleagues from Laser-laboratory Göttingen. It has two channels. All the beams originate from the same supercontinuum 20 MHz pulse laser. The excitation beams are at 570 nm and 650 nm and the STED beams at 710 nm and 745 nm. The setup has three avalanche photodiode detectors, two for dierent channels and one for calibration. One of their researches was focused on astrocyte cells, star shaped brain cells that are involved in synaptic neurotransmissions. They measured the size of vesicles in astrocyte cells. [12] 10

12 The STED at the Joºef Stefan Institute also uses supercontinuum pulse laser. Its pulse repetition is 1 MHz and its pulse width is 150 ps. Its spectrum ranges from blue to infrared. The laser beam is split in to two beams by polarization beam splitter. The excitation and STED pulse have 20 nm wavelength band. The excitation pulse is centered at 532 nm and the STED pulse at 705 nm. All of the research mentioned before used STED setup for the purpose of super-resolution microscopy. This setup however was built to study anisotropic STED eect on dye molecules that are collectively ordered in liquid crystals [12]. The study was conducted by M. Vitek and I. Mu²evi in The liquid crystal used in the experiments was rod like and exhibited smectic-a phase at room temperature, nematic phase at 33 C and isotropic phase at 40 C. It was doped with Nile Red uorescent dye that has the radiative dipole aligned parallel to the molecules of the liquid crystal. The study showed that STED principle allows for very fast (GHz) and ecient control of light by light and has great potential for use in photonic microdevices based on liquid crystals. 4. Conclusion STED microscopy is a far-eld uorescent microscopy technique with the resolution improved by an order of magnitude compared to the diraction limited imaging systems. Its theoretical resolution is unlimited. It is an active eld of research. New improved variations arise simultaneously with the improvements in laser systems. It is fast enough for live specimen imaging and has a resolution, that allows observation of singe molecules such as proteins and DNA molecule. The sample preparation is similar to confocal microscopy. STED is an optical microscopy technique and does not need the samples to be in vacuum and that is one of the reasons it is very popular for imaging of the biological samples. References [1] ( ). [2] S. W. Hell, Microscopy and its focal switch. Nature Methods 6, (2009). [3] T. A. Klar, S. W. Hell, Subdiraction resolution in far-eld uorescence microscopy. Opt. Lett. 24, (1999). [4] J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, S. W. Hell, Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses Opt. Express 19, (2011) [5] S. W. Hell, J. Wichmann, Breaking the diraction resolution limit by stimulated emission: stimulated-emissiondepletion uorescence microscopy. Opt. Lett. 19, (1994). [6] prizes/chemistry/laureates/2014/ ( ). [7] T. S. Tkaczyk, Field guide to microscopy. SPIE, Washington (2009). [8] G. Moneron, R. Medda, B. Hein, A. Giske, V. Westphal, S. W. Hell, Fast STED microscopy with continuous wave ber lasers. Opt. Express 18, (2010). [9] T. A. Klar, E. Engel, S. W. Hell, Breaking Abbe's diraction resolution limit in uorescence microscopy with stimulated depletion beams of various shapes. Phys. Rev. E 64, (2001). [10] D. Wildanger, E. Rittweger, L. Kastrup, S. W. Hell, STED microscopy with a supercontinuum laser source. Opt. Express 16, (2008). [11] ( ). [12] J. Jorga evski, STED mikroskopija. Presented at: Biozikalni seminar, IJS, [13] M. Vitek and I. Mu²evi, Nanosecond control and optical pulse shaping by stimulated emission depletion in a liquid crystal. Opt. Express 23, (2015). [14] ( ). 11